Investigating the response mechanisms of bread wheat mutants to salt stress

Mutation breeding is among the most critical approaches to promoting genetic diversity when genetic diversity is narrowed for a long time using traditional breeding methods. In the current study, 15 wheat mutants created by gamma radiation and three salt-tolerant wheat cultivars were studied under no salinity stress (Karaj) and salinity stress (Yazd) during three consecutive growing seasons from 2017 to 2020 (M05 to M07 generations mutants). Results showed that salinity induced lipid peroxidation and enhanced ion leakage in all genotypes however, M6 and M15 showed the least ion leakage increment. It was also observed that the activity of antioxidant enzymes including SOD, CAT, POX, APX and GR increased with salinity; the maximum increase in antioxidant activity was belonged to M15, M09, M06 and M05. All genotypes had higher protein content in salinity stress conditions; M07 and M12 showed the lowest (1.8%) and the highest (17.3%) protein increase, respectively. Zeleny sedimentation volume increased under salinity stress conditions in all genotypes except M06, C2, C3, and M07. The result indicated that salinity stress increased wet gluten in all genotypes. M10 and M08 showed the highest (47.8%) and the lowest (4%) wet gluten increment, respectively. M06 and M11 mutants showed the lowest (6.1%) and the highest (60.7%) decrement of grain yield due to salinity stress, respectively. Finally, M04, M05, M07, M13, and M14 were known as genotypes with high grain yield in both no salinity and salinity stress conditions. In other word, these genotypes have higher yield stability. The results of the current study revealed that gamma irradiation could effectively be used to induce salinity tolerance in wheat.


Bakery quality
Protein content was significantly affected by region (salinity), genotype, and their interaction (Table 3).All genotypes had higher protein content in salinity stress conditions, although protein increment due to salinity stress was different in studied genotypes (Table 3).M07 and M12 showed the lowest (1.8%) and the highest (17.3%) protein increase, respectively.Zeleny sedimentation volume (%) showed a similar trend to protein content (Table 3), so that, it increased under salinity stress conditions in all genotypes except M06, C2, C3 (no change), and M07 (8% decrease).The bread volume (ml) was increased due to salinity stress in half of the genotypes, whereas it decreased in the other genotypes due to salinity stress (Table 3).The highest increase (24.9%) and the highest decrease (19.7%) of bread volume were observed in M1 and M09, respectively.Region and region × genotype had a significant impact on hardness index, so it increased due to salinity stress in all genotypes except C1 (2.2% decrease) and C3 (no change, Table 3).The result indicated that salinity stress increased wet gluten (%) in all genotypes.M10 and M08 showed the highest (47.8%) and the lowest (4%) wet gluten increment, respectively (Table 3) It was not observed a clear trend in the gluten index of studied genotypes in response to salinity stress.So that its change due to salinity stress ranged from a 76% decrease in M02 to a 67.4% increase in M03.The gluten elasticity of some genotypes was changed as affected by salinity stress (Table 3).It changed from normal to soft in M04, M06, M09, M10, M11, C2, and C3.While, it changed from hard to soft in M02, from normal to hard in M03, and from hard to normal in M08.

Grain yield
The results indicated that the region, genotype, and their interaction had a significant effect on grain yield (Table 4).Grain yield of all genotypes was lower under salinity stress conditions (Yazd) though the reduction due to salinity was not the same in all genotypes (Fig. 1a).M06 and M11 mutants showed the lowest (6.1%) and the highest (60.7%) decrement of grain yield, respectively.Indeed, M11 had the highest grain yield under no salinity stress conditions and the highest grain yield reduction due to salinity stress.As given in Fig. 1a, genotypes are classified into four groups based on comparison of each genotype grain yield in each salinity conditions to average of grain yield of all genotypes in the same salinity conditions; (1) genotypes with high (more than average of  Results also showed a negative and significant correlation between grain yield with protein content, Zeleny sedimentation volume, hardness index, and wet gluten, while the correlation between grain yield and gluten index was positive and significant (Fig. 1b).As given in Fig. 2 grain yield was positively correlated to antioxidant activity and negatively was associated to ion leakage and content of H 2 O 2 and MDA although correlation was greater in salinity stress conditions.In addition, it was observed that there was a positive and significant correlation between ion leakage and content of H 2 O 2 and MDA also there was a positive and significant correlation among enzymatic antioxidants.Clustering the genotypes based on biochemical traits and grain yield in each salinity conditions revealed that genotypes were classified into four groups (Fig. 3).M05, M09, M14, and M15 had the highest grain yield (318 g m -2 on average) and the most antioxidant activity in salinity stress conditions.In no salinity stress conditions, also the maximum grain yield was belonged to C2 and M11 (599 g m -2 on average).

Discussion
In the current study lipid peroxidation (MDA accumulation), H 2 O 2 content and ion leakage were increased due to salinity stress however, their increment were not the same in all genotypes.Salinity stress induces the production of reactive oxygen species (ROS) such as superoxide radical ( O •− 2 ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical ( OH • ) in the plant; ROS are responsible for the peroxidation of membrane lipids and as a result membrane decay and increased ion leakage.In addition, ROS damage other essential macromolecules, photosynthetic pigments, protein, DNA and lipids 23,24 .It has been reported that salt-tolerant genotypes produce smaller amount of ROS rather than salt-sensitive ones 17 or they have a more efficient antioxidant systems 25 .In the current study M02, M06, and M15 had the lowest ion leakage, MDA and H 2 O 2 content increment due to salinity stress and they also a grain yield greater than average grain yield of all genotypes under salinity stress condition nevertheless their grain yield was low in no salinity stress conditions.Therefore, these mutants are suitable for salinity stress conditions or they can be used in plant breeding programs to increase tolerance to salt stress.

Karaj (no salinity stress)
Yazd (salinity stress) Heat map view of studied wheat genotypes based on biochemical traits and grain yield data in no salinity stress (Karaj) and salinity stress (Yazd) conditions (heat map was generated using pheatmap package v. 1.0.12 in R v. 4.2.2).
Our finding showed that activity of different enzymatic antioxidants including SOD, CAT, APX, POX, and GR was increased by salinity stress in all genotypes however, M15, M09, M06, and M05 had a greater antioxidant activity enhancement.7][28][29] .Although ROS play a signaling role in low concentrations when their amount rises due to environmental stress, the plant must scavenge them to reduce oxidative damage to different organelles 29,30 .It has been well documented that different abiotic stresses such as drought and salinity induce oxidative stress in different crops.Plants cope with oxidative stress through triggering antioxidant systems including enzymatic and nonenzymatic antioxidants 31,32 .In addition, our findings showed that POX, CAT, and CAT had the highest increase in activity due to salinity stress, respectively.Several studies have proven that increasing the activity of antioxidant enzymes play an essential role in elevating tolerance to salt stress [33][34][35] .
The salinity stress increased the protein content of all genotypes; M07 and M12 showed the lowest (1.8%) and the highest (17.3%) protein enhancement, respectively.Our results are in accordance with Houshmand et al.,  (2005) who reported that salinity stress increased grain protein of wheat genotypes.It has been reported that salinity stress limits the leaf area index and the ability of the plant to remove dry matter during the grain filling period leading to less starch growing in the grain and then higher protein concentration 37 .Zeleny sedimentation volume (%) showed almost a similar trend to protein content, so that, it increased under salinity stress conditions in all genotypes except M06, control2, control3 (no change), and M07 (8% decrease).Hardness index increased due to salinity stress in all genotypes except control 1 (2.2% decrease) and control 3 (no change).The amount of Zeleny sediment volume describes the degree of sedimentation of the suspended flour in the lactic acid solution over a standard period of time, and this is considered a measure of the quality of the baking.The rate of sedimentation of the flour suspension is affected by the swelling of the gluten part of the flour in the lactic acid solution.Both higher gluten content and better gluten quality result in slower sedimentation and higher values of the Zeleny test.The sedimentation value of flour depends on the protein composition of wheat and is mainly related to the protein content, the hardness of the wheat, and the volume of the pan and hearth loaves 38 .The salinity stress increased wet gluten percentage in all genotypes.M10 and M08 showed the highest (47.8%) and the lowest (4%) wet gluten increment, respectively.Similar to our results, the gluten content of wheat genotypes increased by salinity stress 36 .
Our results showed that the grain yield of all genotypes was significantly higher in no salinity stress conditions.These results are similar to previous studies on the effect of salinity stress on wheat 4,19,37,39,40 .Under salinity stress, high osmotic stress, disruption of nutrient uptake, and ion toxicity cause to reduce cell turgor pressure, limit growth, and decrease grain yield of wheat 41,42 .However, not all of the differences between the two regions were associated with salinity stress.Weather conditions also affected the grain yield of genotypes.As shown in Fig. 4, cumulative precipitation was remarkably higher in Karaj in all growing seasons.Although in the current study, water requirement was met by irrigation, more precipitation certainly has a positive effect on grain yield.The mean temperature during the growing season was 14.7 °C in Karaj and 17.5 °C in Yazd.Also, there were 49 days with a temperature greater than 35 °C in Karaj (about 15 days per growing season), while the number of days with a temperature greater than 35 °C were 95 days (about 31 days per growing seasons).Day numbers with temperatures lower than 0 °C were almost similar for two regions (26 days in Karaj and 29 days in Yazd).This information indicated that in addition to salinity and precipitation, temperature also was more favorable in Karaj.
Mutants showed a different response to salinity stress than controls, so some of them had higher grain yield than control cultivars under salinity stress conditions while the others had an equal to or a lower grain yield than control cultivars.Genetic diversity is an essential prerequisite for developing salt-tolerant wheat genotypes 43 .However, the genetic base of salt-tolerant wheat breeding is narrow, and it limits the progress of salt tolerance in wheat 19 .As shown in the current study, increasing genetic diversity using mutation breeding with gamma irradiation can help to improve salt tolerance in wheat.Likewise, other researchers have used gamma irradiation to increase the genetic diversity of wheat to tolerate salinity stress 44 .

Conclusion
The results of the current study revealed that salinity stress elevated antioxidant activity and decreased grain yield, contrary to baker quality that promoted by salinity stress.In addition, it was found that M05, M09, M14, and M15 had the highest grain yield and the most antioxidant activity in salinity stress.Therefore, these mutants have the potential to be introduced as a new salt-tolerant variety after additional tests in saline areas.

Mutants
In order to evaluate wheat mutants under control conditions (without salinity stress) and salinity stress, this experiment was performed in the form of randomized complete blocks with three replications during 2018, 2019, and 2020 growing seasons.For this experiment, 15 wheat mutants and 3 control cultivars (Arg; C1, Bam; C2, and Narin; C3) were used (Table 5).Control cultivars are originated from temperate and warm regions, and they are relatively salt tolerant 45 .To produce wheat mutants, mutations were made using gamma irradiation with doses of 150 and 200 Gy on Arg and Bam cultivars (Table 6).After that, for several consecutive generations, the mutants were cultivated, and selection was made among them based on their morphological traits and grain yield.Finally, the top 15 mutants were selected for the current study.The fifth to seventh generations (M05 to M07 generations mutants) were planted for this study in 2018, 2019, and 2020 growing seasons, respectively.

Ion leakage
For measuring ion leakage, at the anthesis stage, ten one-cm 2 -piece was taken from flag leaves in each plot and were immersed in distilled water for 20 min at the room temperature.Samples were washed thoroughly then placed in 20 mL of fresh distilled water for 1 h and then the initial electrical conductivity (EC1) was measured.
To measure EC2, the samples were boiled for 5 min, cooled to room temperature and the conductivity was measured again.Ion leakage (IL) was calculated as IL = (EC1/EC2) × 100 53 .

Grain yield
At the time of physiological maturity, after removing the marginal plants, the plants were harvested from three square meters in the center of each plot, and after drying in the open air for a week to equalize the moisture of the samples, threshing was done, and grain yield was measured.

Bakery quality features
Traits related to bakery quality, including protein percentage, Zeleny sediment volume, bread volume, hardness index, wet gluten, gluten elasticity, and gluten index, were measured in the grain chemistry laboratory of Seed and Plant Research Improvement Institute, Karaj, Iran, following the standards of International Association for Cereal Chemistry (ICC).

Statistical design and data analysis
The data homogeneity among different years was evaluated using the Bartlett test.Shapiro-Wilk test was used to evaluate the normality distribution of data.Data were analyzed using the GLM procedure in the SAS environment (SAS 9.4).To do this genotype, and region were considered as fixed factors, and year was considered as a random factor.The least significant difference (LSD) was also used for mean comparison.pheatmap (v.1.0.12) 54, ggally (v.2.1.2) 55 , and corrplot (v.0.92) 56 packages also were used to draw Genotype*Trait, correlation matrix and correlation plot, respectively, in the R (v. 4.2.2) programming environment.

Ethical approval
We confirm that all the experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation.All of the material is owned by the authors and/or no permissions are required.

Figure 1 .
Figure1.(a) Grain yield of studied wheat genotypes in Karaj (without salinity stress) and Yazd (salinity stress) mean data of 2018, 2019, and 2020 years.Red dashed-line; mean grain yield in Karaj (no salinity stress), white dashed-line; mean grain yield in Yazd (salinity stress).Means with the same letter are not significantly different (Slicing method, p < 0.05).(b) Graphic view of pearson correlation matrix between grain yield and and bakery quality characteristics of wheat genotypes (correlation plot was was generated using corrplot package v. 0.92 in R v. 4.2.2).PROT protein content, ZEL Zeleny sedimentation, BV bread volume, HAI hardness index, WGLUT wet gluten, GLUTI gluten index.*, **, and *** significant at p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 4 .
Figure 4. Meteorological data of studied regions during three growing seasons in Karaj (no salinity stress) and Yazd (salinity stress).

Figure 5 .
Figure 5. Location of the study area, A; Alborz (Karaj), Y; Yazd (Ardakan).The points on the map show the coordinates of the experimental farms (Map was generated using ArcMap, ArcGIS, v. 10.6).

Table 2 .
SOD, CAT, POX, APX, and GR of studied wheat genotypes (means and analysis of variance) in Karaj (no salinity stress) and Yazd (salinity stress).

Table 3 .
Characteristics related to bakery quality of wheat genotypes (means and analysis of variance) in Karaj (no salinity stress) and Yazd (salinity stress).For each trait, in each region, means with the same letter are not significantly different (slicing method, p < 0.05).H hard, N normal, S soft.

Table 4 .
Analysis of variance for grain yield of studied wheat genotypes in Karaj (no salinity stress) and Yazd (salinity stress).ns not significant.***Significant at 0.001 probability level.§ Bartlett's test for homogeneity.

Table 5 .
Wheat mutants, their maternal cultivar, and gamma irradiation doses for inducing mutation.

Table 6 .
Control cultivars and their pedigrees.

Table 7 .
Soil and irrigation water properties.